The presence and activity of enzymes can be used to determine the health and metabolic state of a cell. Enzymes are also markers for the cell type since the occurrence and activity of certain enzymes is frequently characteristic of a particular cell. For instance, the activity of certain enzymes can often be used to distinguish cells of bacterial, plant or animal origin. Enzymatic activity and integrity of the cell membrane is one of the criteria for cell viability.
Detection of the presence and activity of enzymes has been facilitated by the development of chromogenic or fluorogenic substrates that are converted by chemical action of the enzyme to a reporter molecule whose optical properties can be measured. Principal among the new reporter molecules have been fluorescent dyes. In many cases the high sensitivity of fluorescence detection permits measurements in living single cells with high spatial and temporal resolution that are not possible with dyes that are not fluorescent.
Unfortunately, many reporter dyes formed by action of intracellular enzymes on synthetic enzyme substrates rapidly leak from the cells, making qualitative and quantitative assays of cellular enzymatic activity difficult. Accurate measurement of enzymatic presence or activity in a single cell requires that the detectable reporter molecule is retained by the cell for the period required for its measurement. Furthermore, it is strongly preferred that the measurement can be made under physiological conditions and, if desired, in living tissue or cells.
An example where these properties are especially required is the use of foreign genes that code for enzymes as markers in cells. For instance, bacterial genes that code for production of β-galactosidase (lacZ) or β-glucuronidase (GUS) have been incorporated (transletted) (transfected)into plant and animal cells that do not ordinarily contain this activity. To determine the success of the incorporation, to sort and clone the transletted transfected cells, and to measure the effect of certain promoter genes that are simultaneously incorporated with the marker gene requires both that the reporter molecule formed by action of the enzyme on the substrate is retained in the single cell, and that its retention measures the level of enzymatic activity in the cell. The analysis and sorting of successfully cloned cells can be performed by flow cytometry if suitable probes are available that are retained inside the cell sufficiently long for the analysis to be completed.
A number of synthetic enzyme substrates have been used to determine the activity of enzymes in extracts of cells, in solution, or in living cells. Most of these substrates have been based on fluorescent reporter dyes such as 7-hydroxy-4-methylcoumarin (β-methylumbelliferone), 7-amino-4-methylcoumarin, 4-methoxy-2-naphthylamine, fluorescein, resorufin, rhodamine 110 or various derivatives of α- and β-naphthol. Quantitative detection of enzymatic activity in single, living cells under physiological conditions, whether intrinsic to the cell or incorporated by genetic manipulation, has been difficult because the reporter molecules formed by the enzymatic reaction of substrates based on the above fluorophores have been poorly retained in cells.
Certain substrates have been described for the histochemical localization of protease enzymes in cultured cells, that require addition of a second reagent to develop a weakly fluorescenct fluorescent product subsequent to the enzymatic reaction by Gossrau, Cytochemistry of Membrane Protease, HISTOCHEM. J. 17, 737 (1985). The requirement of a second reagent complicates the quantitative aspects of the measurements and makes the Gossrau procedure less desirable for use in applications that are highly automated.
Use offluorescein of fluorescein digalactoside (FDG) in an automated procedure to sort single lacZ-positive transfected cells has been described by Nolan et al., MONITORING OF CELLS AND TRANS-ACTING TRANSCRIPTION ELEMENTS, U.S. Pat. No. 5,070,012 (1991). Cells were loaded with this relatively impermeant substrate by hypoosmotic shock, then the cells were cooled to below 5° C. Although this permitted sorting of the cells at a super-cooled temperature where the competition between the enzyme turnover rate and the leakage rate permitted retention of a portion of the fluorescent dye, it is not effective under physiological conditions. The leakage rate of potential alternative probes for monitoring lacZ or GUS gene fusion such as the resorufin formed by hydrolysis of resorufin galactoside and β-methylumbelliferone formed by hydrolysis of β-methylumbelliferyl glucuronide tends to be even faster.
Lipophilic derivatives of fluorescein and resorufin have been described in LIPOPHILIC FLUORESCENT GLYCOSIDASE SUBSTRATES (U.S. Pat. No. 5,208,148 to Haugland et al. (1993)) and LONG WAVELENGTH LIPOPHILIC FLUOROGENIC GLYCOSIDASE SUBSTRATES (U.S. Pat. No. 5,242,805 to Naleway et al. (1993)), where retention is enhanced by the addition of a lipophilic residue and can be used under physiological conditions. These substrates can be used with automated procedures but are not effective where the fluorescent label needs to be retained inside cells to which a fixative has been applied.
There is a need for a method for analyzing metabolic activity in cells under physiological conditions, using a substrate that yields fluorescence or absorbance detectable products that are retained in the cells for the time period required to accomplish relevant analysis. The fluorescence or absorbance should be detectable inside fixed cells as well as living cells and be distinct from background fluorescence or absorbance.
Glutathione transferase is known to catalyze the reaction of a wide variety of alkylating and arylating reagents with glutathione (Mantle, et al., Glutathione S-transferases, BIOCHEM. SOC. TRANSACTIONS, 18, 175 (1990)). This is an important process for detoxification of pollutants and dangerous chemicals by cells. The enzyme is ubiquitous, being found in both plant and animal cells. Free glutathione levels in most normal cells are relatively high (up to >1 mM).
A number of haloalkylated fluorescent reagents have been previously described for use in estimating the level of intracellular thiols in cells. Monochlorobimane is a chloroalkylated fluorescent reagent that has been used to measure glutathione levels in single cells by flow cytometry in a reaction that is catalyzed by glutathione transferase (Rice, et al., Quantitative Analysis of Cellular Glutathione by Flow Cytometry Utilizing Monochlorobimane: Some Applications to Radiation and Drug Resistance in Vitro and in Vivo, CANCER RESEARCH 46, 6105 (1986)). Monobromobimane is a reagent with similar utilizing however it is less selective for intracellular thiols than is monochlorobimane (Fernández-Checa, et al., The Use of Monochlorobimane to Determine Hepatic GSH Levels and Synthesis, ANALYT. BIOCHEM. 190, 212 (1990)). The use of chloromethylfluorescein diacetate (CMFDA) and chloromethyleosin diacetate (CMEDA) has previously been described for measuring the content of thiols in living cells during the cell cycle by flow cytometry, by Poot, et al., Flow Cytometric Analysis of Cell Cycle-Dependent Changes in Cell Thiol Level by Combining a New Laser Dye With Hoechst 33342, CYTOMETRY 12, 184 (1991). Although the two probes described by Poot, et al. may prove useful for this invention, the specific haloalkylated reagents of Poot et al. were not utilized to detect or quantitate the presence or activity of a second enzyme, other than glutathione transferase, in live or fixed cells or to determine the viability of the cell.
Mangel et al. has described a family of fluorogenic protease substrates having amino acids or peptides bound to the amino nitrogens of a rhodamine fluorophore (U.S. Pat. Nos. 4,557,862 (1985) and 4,649,893 (1987)). Upon cleavage of the amino acids by proteases, the rhodamine becomes fluorescent. However, the Mangel et al. compounds do not possess a haloalkyl moiety to aid in retaining the fluorescent product within cells, and typically leak from cells fairly quickly after enzyme cleavage. The Mangel et al. compounds are also generally difficult to load into cells. This is primarily due to the size of the substrates, but can also be due to the presence of highly polar amino acid residues used as blocking groups. Also, the need for the enzyme to cleave two blocking groups from the substrate complicates the kinetics of the enzyme reaction considerably, as cleavage of the second fluorophore-amino acid bond is much slower than cleavage of the first. Finally, the preparation of the Mangel et al. substrates requires two equivalents of the relatively expensive target peptides for each fluorophore so labeled.
The presence of the haloalkyl group is critical for retention of the reporter molecule in cells. In a direct comparison of a reporter molecule and its chloromethyl-substituted analog, it was found that the chloromethyl-substituted reporter was retained in cells for several hours, while the conventional reporter leaked away quickly (Examples 15 and 16). Similarly, the nature of the haloalkyl group itself is critical. While haloalkyl-substituted hydrolase substrates based on coumarin fluorophores have been described by Scheper et al., (U.S. Pat. No. 4,777,269 (1988)), Scheper et al.'s preferred haloalkyl substituent, trifluoromethyl, does not aid in retaining the reporter molecular within cells. A comparison of cellular retention based on haloalkyl group was performed (Example 16), and verified that the chloromethyl-substituted reporter was well-retained in cells, while the methyl- and trifluoromethyl-substituted reporters show very poor retention.